Method of retrovirus storage

ABSTRACT

A method of retrovirus storage, characterized in that a retrovirus is sustained in the presence of a substance with retrovirus binding activity immobilized on a solid phase. Further, there is provided a retrovirus composition characterized in that a retrovirus in the form of binding to a substance with retrovirus binding activity is sealed in a container holding a solid phase having the substance with retrovirus binding activity immobilized thereon.

TECHNICAL FIELD

The present invention relates to a method for purifying or storing aretrovirus vector used for transforming a cell by gene transfer in thefields of medicine, pharmacy, agriculture, forestry and fisheries aswell as food science, and a series of techniques related thereto.

BACKGROUND ART

Retrovirus vectors can be used to surely integrate genes of interestinto host chromosomes. For this reason, these vectors are widely used inthe field of gene therapy centering on ex vivo gene therapy protocolsthat target hematopoietic stem cells or peripheral blood lymphocytes.They also are widely used in the field of basic research as tools forgene expression analyses. This is because they can be used to achievestable expression levels of foreign (inserted) genes. Usually, a culturesupernatant of a producer cell is filtrated through a filter and thefiltrate is used as a retrovirus vector. The filtrate may be subjectedto further purification if the retrovirus vector is to be used for an exvivo gene therapy protocol. However, there have been problems concerningthe above because the purification of a virus vector is complicated andthe recovery is low. No scientific literature describing the stabilityof a purified virus vector is found.

A culture supernatant of a retrovirus vector producer cell is usuallystored after filtration through a filter in a frozen state in a deepfreezer. The stability of a thawed vector in a solution state is low.The half-life has been reported to be 92 hours at 4° C., 18-64 hours at0° C., 11-39 hours at 32° C., or 7-9 hours at 37° C. (Non-patentDocument 1, Non-patent Document 2). Since the stability of a retrovirusvector is low as described above, a cryopreserved retrovirus vector isusually thawed upon use and used immediately.

A method in which a recombinant retrovirus is lyophilized and thenstored is described in Patent Document 1. This method requires equipmentfor lyophilization, and a procedure for reconstituting the storedrecombinant retrovirus upon use.

A gene transfer method in which a retrovirus is subjected to infectionin the presence of a substance having a retrovirus-binding activity (inparticular, a fibronectin fragment) is described in Patent Document 2,Patent Document 3 or Non-patent Document 3. The influence of such asubstance on the stability of a retrovirus is unknown.

Patent Document 1: U.S. Pat. No. 5,792,643

Patent Document 2: WO 95/26200

Patent Document 3: WO 97/18318

Non-patent Document 1: McTaggart, S., Al-Rubeai, M., Biotechnol. Prog.,16(5):859-865 (2000)

Non-patent Document 2: Kaptein, L. C., et al., Gene Ther., 4(2):172-176(1997)

Non-patent Document 3: Hanenberg, H. et al., Nat. Med., 2(8):876-882(1996)

DISCLOSURE OF INVENTION Problems to be Solved by the Invention

As described above, one must rapidly handle a cryopreserved retrovirusvector after thawing it and use to infect a cell in a short time. Thus,if the schedule of infection step is upset for some reason (e.g.,delayed growth of a cell to be infected with a retrovirus vector) and aretrovirus vector cannot be used to infect a cell immediately afterthawing, sufficient infection efficiency may not be achieved.

Thus, a method by which a retrovirus vector can be stably stored in astate ready for immediate use has been desired.

Means to Solve the Problems

The present inventors attempted to develop a method for stably storing aretrovirus at a low temperature without freezing while retaining thegene transfer activity. As a result of intensive studies, the presentinventors have found that a retrovirus can be stably stored in a stateready for immediate use even in an unfrozen state by maintaining theretrovirus at a low temperature being bound to a solid support coatedwith a substance having a virus-binding activity. Thus, the presentinvention has been completed.

The first aspect of the present invention relates to a method forstoring a retrovirus, the method comprising maintaining a retrovirus inthe presence of a substance having a retrovirus-binding activityimmobilized on a solid support.

According to the first aspect, the retrovirus can be maintained in anunfrozen state. For example, the retrovirus may be maintained in asolution so that the solution is not in contact with air in oneembodiment of the storage method.

According to the first aspect, the retrovirus may be separated fromother retrovirus producer cell-derived components. For example, theretrovirus can be maintained in a buffer containing a phosphate salt asa buffering component in this embodiment. In addition, the retrovirusmay be maintained using as the buffer a solution containing a substanceselected from the group consisting of proteins and saccharides.

Examples of the substances having a retrovirus-binding activity usedaccording to the method for storing a retrovirus of the first aspectinclude a polypeptide having the heparin-II domain of fibronectin,fibroblast growth factor, a polypeptide having the insulin-bindingdomain of type V collagen, DEAE-dextran and polylysine.

The second aspect of the present invention relates to a compositioncontaining a retrovirus, wherein the composition is contained in acontainer carrying a solid support on which a substance having aretrovirus-binding activity is immobilized, and the retrovirus is boundto the substance having a retrovirus-binding activity.

For example, the retrovirus in the composition of the second aspect maybe contained in a solution in the container so that the solution is notin contact with air.

In the composition of the second aspect, the retrovirus may be containedbeing separated from other retrovirus producer cell-derived components.The composition can contain a buffer containing a phosphate salt as abuffering component. In addition, the composition may contain asubstance selected from the group consisting of proteins and saccharidesin a solution.

Examples of the substances having a retrovirus-binding activity used forstoring a retrovirus according to the second aspect include apolypeptide having the heparin-II domain of fibronectin, fibroblastgrowth factor, a polypeptide having the insulin-binding domain of type Vcollagen, DEAE-dextran and polylysine.

EFFECTS OF THE INVENTION

According to the present invention, a retrovirus can be stored so thatit can be immediately subjected to gene transfer. It is possible toconduct gene transfer with reproducibility because retrovirus-boundcontainers of the same quality can be stably stored. Furthermore, thepresent invention enables transportation at a low temperature if agas-permeable cell culture bag or separation bag is used as a storagecontainer to keep a closed system.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows values of gene transfer efficiency relative to transferefficiency observed for the control.

FIG. 2 shows values of gene transfer efficiency relative to transferefficiency observed for the control.

BEST MODE FOR CARRYING OUT THE INVENTION

There is no specific limitation concerning the retrovirus used accordingto the present invention. For gene transfer, an artificially modifiedrecombinant retrovirus (i.e., a retrovirus vector) is usually usedaccording to the present invention. Particularly, areplication-defective retrovirus vector is preferable for preventingunlimited infection or gene transfer. Such a vector is madereplication-defective so that it cannot autonomously replicate in aninfected cell and therefore avirulent. Such a vector can invade a hostcell such as a vertebrate cell (particularly a mammalian cell) to stablyintegrate a foreign gene, which is inserted in the vector, into thechromosomal DNA. Examples of known replication-defective retrovirusvectors include retrovirus vectors (e.g., MFG vector, α-SGC vector (WO92/07943), pBabe (Morgenstern, J. P., Land, H., Nucleic Acids Research,18(12):3587-3596 (1990)), pLXIN (Clontech) or pDON-AI (Takara Bio)),lentivirus vectors (human immunodeficiency virus (HIV)-derived vectors,simian immunodeficiency virus (SIV)-derived vectors, etc.) andmodifications thereof.

There is no specific limitation concerning the foreign gene carried bythe retrovirus vector. Any gene of which the expression in the cell ofinterest is desired can be inserted. Examples thereof include genesencoding polypeptides (enzymes, growth factors, cytokines, receptors,structural proteins, etc.), antisense RNAs, ribozymes, decoys, and RNAsthat cause RNA interference. It is possible according to the presentinvention to use the foreign gene being inserted into a retrovirusvector under the control of an appropriate promoter (e.g., an LTRpromoter in the retrovirus vector or a foreign promoter). Anotherregulatory element which cooperates with the promoter and atranscription initiation site (e.g., an enhancer sequence) may bepresent in the vector in order to accomplish transcription of theforeign gene. Preferably, the transferred gene may contain a terminatorsequence placed downstream. Furthermore, one may include an appropriatemarker gene which enables selection of a cell having a transferred gene(e.g., a drug resistance gene, a gene encoding a fluorescent protein, agene encoding an enzyme that can function as a reporter such asβ-galactosidase or luciferase).

The retrovirus may be prepared according to a known method and usedaccording to the present invention. There is no specific limitationconcerning the preparation method. If a retrovirus vector is to be used,a culture supernatant collected from a culture of a retrovirus producercell suitable for the retrovirus can be used according to the presentinvention. The retrovirus producer cell may be one that stably producesretrovirus particles in the supernatant or one that transiently producesretrovirus particles upon transfection with a retrovirus vector plasmid.

A known packaging cell line such as PG13 (ATCC CRL-10686), PA317 (ATCCCRL-9078), GP+E-86 or GP+envAm-12 (U.S. Pat. No. 5,278,056), or Psi-Crip(Danos, O., Mulligan, R. C., Proc. Natl. Acad. Sci. USA,85(17):6460-6464 (1988)) may be used for preparing a retrovirus producercell. 293 cell or 293T cell of which the transfection efficiency is highmay be used for preparing a retrovirus producer cell.

According to the present invention, it is also possible to use aretrovirus prepared by pseudotyped packaging which has an envelopederived from a virus different from the one from which the genome of theretrovirus vector is derived. For example, a pseudotyped retrovirushaving an envelope derived from Moloney murine leukemia virus (MoMLV),gibbon ape leukemia virus (GaLV), vesicular stomatitis virus (VSV) orfeline endogenous virus, or a protein that can function as an envelopecan be used. Furthermore, one may prepare a retrovirus vector having onits surface a protein that is subjected to sugar chain modification. Theretrovirus vector may be prepared using a retrovirus producer cellhaving a transferred gene for an enzyme involved in glycosylation or thelike. Such a retrovirus vector can also be used according to the presentinvention.

(A) The Method for Storing a Retrovirus of the Present Invention

The present invention provides a method for storing a retrovirus, themethod comprising maintaining a retrovirus in the presence of asubstance having a retrovirus-binding activity immobilized on a solidsupport in an unfrozen state.

There is no specific limitation concerning the container for storing aretrovirus used according to the method of the present invention as longas it is suitable for storage of a biological material (e.g., a cell ora body fluid sample) or can be used to culture cells. Examples thereofinclude culture plates, culture flasks, separation bags andgas-permeable culture bags.

According to the present invention, there is no specific limitationconcerning the solid support on which a substance having aretrovirus-binding activity is to be immobilized. Solid supports invarious forms including beads and fibers can be used. It is preferablefor the present invention to use a solid support made of a material thatdoes not have a harmful influence on maintenance or growth of cells whenit is brought into contact with the cells during cell cultivation. Inone preferred embodiment, the above-mentioned container for storing aretrovirus is used as a solid support for immobilizing a substancehaving a retrovirus-binding activity. In this embodiment, the surface ofthe container to be brought into contact with the content is coated witha substance having a retrovirus-binding activity. Examples of substanceshaving a retrovirus-binding activity include fibronectin, a fibronectinfragment having the heparin-II domain (CH-296 (RetroNectin), CH-271,H-296, etc.), fibroblast growth factor, a polypeptide having theinsulin-binding domain of type V collagen, DEAE-dextran and polylysine.There is no specific limitation concerning the method for immobilizing asubstance having a retrovirus-binding activity on the surface of a solidsupport. A method suitable for the substance having a retrovirus-bindingactivity to be used may be selected. In an exemplary method, a buffercontaining the substance is allowed to stand for a given period of timebeing in contact with a solid support to be used. Procedures forimmobilizing the substance are also described in Patent Documents 2 and3.

Although it is not intended to limit the present invention, it ispreferable according to the present invention to reduce the amount ofair that is in contact with a retrovirus-containing solution. A methodin which a container to be used has a structure with which the solutioncan be held so that the solution is not in contact with air exemplifiesone embodiment of the present invention. If a container of a fixedvolume is to be used, this embodiment can be accomplished by filling thecontainer with the retrovirus-containing solution. Alternatively, asolution of an arbitrary volume can be held so that the solution is notin contact with air. This is accomplished by using a container in a formof sack or bag composed of a film-like substrate of which the internalvolume can be altered according to the volume of the solution. Thecontainer used according to the present invention preferably can hold aretrovirus-containing solution in a sealed or airtight state. Morepreferably, a commercially available cell culture bag can be usedaccording to the present invention.

A supernatant collected using a retrovirus producer cell, a retroviruspurified from the supernatant or the like can be used as a retrovirusstored according to the present invention. For example, a supernatant ora purified retrovirus which was cryopreserved before may be stored in anunfrozen state according to the method of the present invention.

In one embodiment of the present invention, a retrovirus is stored beingseparated from other retrovirus producer cell-derived components. Thisembodiment is usually carried out with the following steps:

(1) a step of contacting a solid support on which a substance having aretrovirus-binding activity is immobilized with a retrovirus producercell culture supernatant which contains the retrovirus;

(2) a step of washing the solid support of step (1); and

(3) a step of maintaining the solid support obtained in step (2) beingin contact with a buffer.

If a retrovirus that has been purified beforehand is to be used in placeof a supernatant of a retrovirus producer cell, step (2) may be omitted.

In this embodiment, the infectivity of a retrovirus is retained at ahigher level because inactivation of the retrovirus due to a componentcontained in the supernatant of the retrovirus producer cell issuppressed.

There is no specific limitation concerning the method for binding aretrovirus to a substance having a retrovirus-binding activity. Forexample, the binding may be carried out as follows: a culturesupernatant of a retrovirus producer cell is allowed to stand being incontact with a solid support on which the substance is immobilized; aretrovirus is precipitated on the surface of a solid support usingcentrifugal force; or a container containing a solid support and aretrovirus is shaken.

A supernatant is removed from a container to which a retrovirus is boundvia a retrovirus-binding substance as a result of the above-mentionedprocedure, the solid support (e.g., the container itself) is optionallywashed, and a solution suitable for storage of the retrovirus is thenadded to the container.

There is no specific limitation concerning the solution used for washinga solid support to which a retrovirus is bound as long as it does notconsiderably reduce the infectivity of the retrovirus to be stored. Forexample, physiological saline, phosphate-buffered saline or the samemedium as that used for culturing the retrovirus producer cell can beused. In particular, a solution used for storing a retrovirus asdescribed below is preferably used. Since a supernatant of a retrovirusproducer cell contains a substance that inhibits infection of a cellwith a retrovirus, a retrovirus stored being separated from otherretrovirus producer cell-derived components is advantageously used toinfect a cell.

According to the present invention, a retrovirus is stored being boundto a retrovirus-binding substance and in contact with an appropriatesolution. Although there is no specific limitation concerning thesolution to be used in this step as long as it does not considerablyreduce the infectivity of the retrovirus to be stored, a buffercontaining a phosphate salt (sodium phosphate, potassium phosphate,etc.) as a buffering component is preferably used. The solution mayfurther contain a saccharide (glucose, galactose, lactose, mannitol,etc.), a protein (albumin, collagen (gelatin), etc.) or anothercomponent (an inorganic salt, a polyol, human serum, etc.) as acomponent for stabilizing a retrovirus.

A retrovirus is stored in an unfrozen state according to theabove-mentioned storage method. Nevertheless, the ability of the storedretrovirus to infect a cell is retained at a higher level than thatobserved for a retrovirus culture supernatant stored as it is. Althoughit is not intended to limit the present invention, a retrovirus isstored according to the present invention at a low temperature for aperiod of 24 hours or more, preferably 48 hours or more, more preferably72 hours or more. As used herein, a low temperature refers to atemperature of 15° C. or below at which a solution to be stored does notfreeze. Preferably, a retrovirus is stored at 0-10° C.

A retrovirus stored in a container according to the method of thepresent invention can be used for infection as it is. If the retrovirusis a recombinant retrovirus carrying a foreign gene, it is possible totransfer the gene into a cell by this procedure. For example, infectionwith a retrovirus can be carried out by adding to a container a cell ofwhich the infection is desired after exchanging the solution in thecontainer for a solution suitable for infection of the cell with theretrovirus (or without the exchange if the solution for storage issuitable for infection with the retrovirus). Alternatively, infectionwith a retrovirus can be carried out by adding a cell suspension to astorage container from which the contained solution has been removed.The infection step may be carried out in static culture. Alternatively,the infection step may be carried out according to a method in which aretrovirus is brought into contact with a cell by applying centrifugalforce to precipitate the cell on the surface of a solid support on whicha substance having a retrovirus-binding activity is immobilized. Thetarget cell infected with the retrovirus may be cultured in thecontainer as it is, or it may be transferred to another container afterthe above-mentioned procedure and then cultured.

If the retrovirus-binding substance also has an affinity for a targetcell, the target cell and the retrovirus are co-localized on the surfaceof the container as a result of the above-mentioned procedure. Then,infection of the target cell with the retrovirus takes place with highefficiency. For example, gene transfer into a hematopoietic stem cellcan be conveniently carried out with high efficiency by using arecombinant retrovirus carrying a foreign gene of interest and thepolypeptide CH-296, which has a hematopoietic stem cell-bindingactivity.

A retrovirus storage method in which a solid support on which both asubstance having a retrovirus-binding activity and a substance having atarget cell-binding activity are immobilized is used exemplifies anotherembodiment of the present invention. According to this method, acontainer in which a retrovirus is stored together with a substancehaving a retrovirus-binding activity and a substance having a targetcell-binding activity can be used as a container for infection of atarget cell with a retrovirus. Thus, the method is particularly usefulin the field of gene therapy for which highly efficient and/orcell-selective gene transfer is desired.

Examples of substances having a target cell-binding activity used in theabove-mentioned embodiment include, but are not limited to, a protein ora peptide that is capable of recognizing a target cell (an antibody or areceptor that recognizes a component on the surface of a target cell, aligand for a receptor on the surface of a target cell (a growth factor,a hormone, a cytokine, etc.)), a lectin, a sugar chain and a glycolipid.Such substances and methods for immobilizing the same are also describedin Non-patent Document 3.

(B) The Composition of the Present Invention

The present invention provides a retrovirus-containing composition whichis in a form suitable for storage.

A retrovirus in the composition is contained in a container being boundto a substance having a retrovirus-binding activity immobilized on asolid support. The composition can be prepared in accordance with theabove description regarding the retrovirus storage method. The substancehaving a retrovirus-binding activity is exemplified by theabove-mentioned one that can be used according to the retrovirus storagemethod.

In one embodiment of the present invention, an exemplary compositioncontains a retrovirus in a solution so that the solution is not incontact with air. The composition may be separated from other retrovirusproducer cell-derived components. In this case, the compositionpreferably further contains a solution suitable for storage of aretrovirus. The above-mentioned solution that can be used according tothe retrovirus storage method may be used as the solution suitable forstorage of a retrovirus.

Although there is no specific limitation concerning the container usedaccording to the present invention as long as it is suitable for storageof a biological material (e.g., a cell or a body fluid sample) or can beused to culture cells, the container preferably can hold aretrovirus-containing solution in a sealed or airtight state. Forexample, a commercially available cell culture bag can be used.

The retrovirus-containing composition of the present invention hasexcellent storage stability and can be immediately used to infect a cellwith a retrovirus. Thus, it is useful for studies of retroviruses and inthe field of medicine, particularly in the field of gene therapy inwhich recombinant retrovirus vectors are used.

EXAMPLES

The following Examples illustrate the present invention in more detail,but are not to be construed to limit the scope thereof.

Example 1 1. Preparation of CH-296-Coated Plate

500 μl of a fibronectin fragment CH-296 (product name: RetroNectin;Takara Bio) at a concentration of 20 μg/ml was added to each well of a24-well plate without surface treatment (Falcon). The plate was allowedto stand at 4° C. overnight, subjected to blocking with 2% BSA/PBS atroom temperature for 30 minutes, and washed with PBS. This plate wasused as a CH-296-coated plate and prepared when necessary.

2. Preparation of Retrovirus Vector

A retrovirus vector plasmid pDOG-polII was constructed as follows.First, an rsGFP expression vector pQBI25 (Qbiogene) was cleaved withrestriction enzymes NheI and NotI to obtain a 775-bp GFP gene fragment.Next, pQBI polII (Qbiogene) was cleaved with restriction enzymes NheIand NotI to remove an rsGFP-NeoR fusion gene. The previously obtained775-bp rsGFP gene fragment was inserted to obtain a vector pQBIpolII(neo−) in which the rsGFP gene is expressed under the control ofpolII promoter. pQBI polII(neo−) was digested with a restriction enzymeXhoI to obtain a DNA fragment containing a GFP expression unit under thecontrol of polII promoter. The termini were blunted using DNA BluntingKit (Takara Bio). Termini of a 4.58-kbp vector fragment obtained bydigesting a retrovirus vector plasmid PDON-AI (Takara Bio) withrestriction enzymes XhoI and SphI were blunted using DNA Blunting Kit(Takara Bio), and then dephosphorylated using alkaline phosphatase(Takara Bio). The previously blunted DNA fragment containing the rsGFPexpression unit under the control of polII promoter was inserted intothis blunted vector using DNA Ligation Kit (Takara Bio) to obtain anrsGFP expression recombinant retrovirus vector pDOG-polII.

Transient virus production was carried out using the vector pDOG-polIIand Retrovirus Packaging Kit Eco (Takara Bio) to obtain an ecotropicvirus DOG-polII. The ecotropic virus DOG-polII was used to infect a GaLVretrovirus packaging cell PG13 (ATCC CRL-10686) in the presence ofRetroNectin (Takara Bio) to obtain a gene-transferred cellPG13/DOG-polII. PG13/DOG-polII was cultured in Dulbecco's modified Eaglemedium (DMEM, Sigma) containing 10% fetal calf serum (Thermo Trace).When the cell was grown to semi-confluence, the medium was exchanged for0.1 ml/cm² of fresh DMEM containing 10% fetal calf serum. After 24hours, the supernatant was filtrated through a 0.45-μm filter(Millipore) to obtain a GaLV/DOG-polII virus supernatant. Aliquots ofthe thus obtained virus supernatant were stored in a freezer at −80° C.and subjected to subsequent storage and gene transfer experiments.

3. Measurement of Titer of Virus Supernatant

A titer of a virus supernatant was measured using HT-1080 cells (ATCCCCL-121) according to a standard method (Markowitz, D. et al., J.Virol., 62(4):1120-1124 (1988)). Specifically, 5×10⁴ HT-1080 cells in 2ml of DMEM containing 10% fetal calf serum were added to each well of a6-well tissue culture plate, and cultured overnight at 37° C. with 5%CO₂. The medium was removed by suction, 1 ml of a serial dilution of thevirus supernatant was added to the well, and hexadimethrine bromide(polybrene, Aldrich) at a final concentration of 8 μg/ml was furtheradded thereto. The cells were cultured at 37° C. with 5% CO₂ for 4 to 6hours. 1 ml of DMEM containing 10% fetal calf serum was further addedthereto, and the cultivation was continues for 72 hours. The cellscollected from the plate were subjected to analysis using a flowcytometer FACS Vantage (Becton-Dickinson), and the ratio of HT-1080cells expressing rsGFP was determined. The number of infectiousparticles in 1 ml of a supernatant (I.V.P./ml) was calculated based on avalue obtained by multiplying the number of input cells per well by theratio of rsGFP-expressing cells and the dilution rate of the virussupernatant to determine the virus titer. The titers of virussupernatant prepared in Example 1-2 ranged from 1.9×10⁵ I.V.P./ml to4.5×10⁵ I.V.P./ml.

Example 2

Storage of Retrovirus Using CH-296-Coated Plate (1)

K-562 cells (ATCC CCL-243) were used for assessment of the activity ofthe GaLV/DOG-polII virus supernatant. K-562 cells were cultured inRPMI-1640 medium (Sigma) containing 10% fetal calf serum (Thermo Trace).250 μl of the GaLV/DOG-polII virus supernatant (the originalconcentration) was added to each well of a 24-well CH-296-coated plate,and the plate was incubated in 5% CO₂ incubator at 37° C. for 4 hours tobind the retrovirus vector. Each well was washed twice with 500 μl ofPBS (phosphate-buffered saline), 0.1% bovine serum albumin (BSA,Fraction V, Sigma) in PBS, 1% bovine serum albumin in PBS, X-VIVO 15(Cambrex), X-VIVO 10 (Cambrex), IMDM (Invitrogen) or RPMI1640 (Sigma).The well was filled with 500 μl of the same solution, and the plate wasincubated at 4° C. for 7 days. In addition, the virus supernatant wasadded to each well, and the plate was incubated for 7 days withoutwashing (the virus supernatant was incubated as it was). On day 7, thesolution was removed, 500 μl of a suspension containing K-562 cells at adensity of 4×10⁴ cells/ml was added, and the cells were cultured in thepresence of 5% CO₂ at 37° C. for gene transfer. As a control, avirus-bound plate was prepared using a virus supernatant of the same lotand the same procedure, and K-562 cells were added immediately withoutincubation at 4° C. for gene transfer. Gene transfer efficiency wasdetermined for the cells using expression of rsGFP gene as an index.Values of gene transfer efficiency with the virus relative to transferefficiency observed for the control after incubation for 7 days areshown in FIG. 1.

As shown in FIG. 1, higher activities were retained using all thesolutions as compared with that observed for the virus supernatant thatwas allowed to stand as it was. Thus, it was shown that storage of aretrovirus being separated from a supernatant of a retrovirus producercell was effective. In particular, a solution containing bovine serumalbumin proved to be effective in stable storage.

Example 3 Storage of Retrovirus Using CH-296-Coated Plate (2)

It was shown in Example 2 that storage stability was increased bywashing a retrovirus-bound container. Next, influence of the followingsubstances on storage of a retrovirus was examined in order to searchfor a solution that can contribute more to increase in storagestability: inorganic salts (sodium phosphate; a mixture of calciumchloride and magnesium sulfate; a mixture of sodium phosphate, calciumchloride and magnesium sulfate); saccharides (glucose; D-sorbitol;refined white sugar; maltose; fructose; lactose; D-mannitol);macromolecular compounds (carmellose sodium; methylcellulose;hydroxypropyl cellulose; propylene glycol; polyethylene glycol 400);proteins (purified gelatin; human serum albumin (HSA)). Among theabove-mentioned substances, inorganic salts were dissolved in water forinjection and other substances were dissolved in PBS. Then, theireffects on retrovirus storage stability were tested. The procedure forthe search followed that of Example 2 except that the storage incubationof a retrovirus-bound container was carried out at 37° C. for 3 hours.As a result, increase in retrovirus vector storage stability wasobserved using the following as compared with PBS alone: 40 mM sodiumphosphate buffer (pH 7); 0.5 mM calcium chloride and 1 nM magnesiumsulfate in 20 mM phosphate buffer (pH 7); 1.5-20% human serum albumin inPBS; 0.5-2.5% purified gelatin in PBS; or 5% lactose in PBS.

As a result of the above-mentioned tests, it was shown that there was atendency that 40 mM sodium phosphate buffer was superior to PBS as abasal solvent, and proteins and saccharides increased the stability.Then, retrovirus storage stability tests were carried out at 4° C. for 7days according to the procedure as described in Example 2 using thefollowing: 1% bovine serum albumin in PBS; 1.5% human serum albumin in40 mM sodium phosphate buffer; 0.5% purified gelatin in 40 mM sodiumphosphate buffer; or 5% lactose in 40 mM sodium phosphate buffer. pH ofthe sodium phosphate buffer was 7.0 in all cases. Values of genetransfer efficiency with the virus relative to transfer efficiencyobserved for the control after incubation for 7 days were calculated andare shown in FIG. 2. As shown in the figure, it was shown that thestorage stability was dramatically increased by storage after exchangefor the respective solutions as compared with the storage of theretrovirus producer cell supernatant as it was.

Example 4 Storage of Retrovirus in the Presence of Various FunctionalSubstances

In this Example, storage stability was assessed using a functionalsubstance that exhibits a retrovirus-binding activity and a celladhesion activity. Besides CH-296, the following was used as afunctional substance that exhibits a retrovirus-binding activity and acell adhesion activity: CH-271 or H-296 (Kimizuka, F. et al., J.Biochem., 110(2):284-291 (1991)); DEAE-dextran (Sigma); poly-L-lysine(average molecular weight of 30,000 to 70,000, ICN); a mixture ofDEAE-dextran and a fibronectin fragment C-CS1 (Kimizuka, F. et al., J.Biochem., 110(2):284-291 (1991)); or a mixture of poly-L-lysine andC-CS1.

500 μl of a fibronectin fragment CH-296, CH-271 or H-296 (20 μg/ml),DEAE-dextran in PBS (0.9 mg/ml), or poly-L-lysine in PBS (40 μg/ml) wasadded to each well of a 24-well plate without surface treatment(Falcon). The mixture of DEAE-dextran and C-CS1 and the mixture ofpoly-L-lysine and C-CS1 were prepared by adding C-CS1 to theDEAE-dextran solution or the poly-L-lysine solution at a finalconcentration of 4.4 μg/ml. The plate was allowed to stand at 4° C.overnight, subjected to blocking with 2% BSA/PBS at room temperature for30 minutes, and washed with PBS. This plate was used as a functionalsubstance-coated plate and subjected to retrovirus storage stabilitytests.

Retrovirus storage stability tests were carried out using the platecoated with the respective functional substances according to theprocedure as described in Example 2. The tests were carried out in thefollowing three virus storage manners: storage of the virus supernatantas it was; storage in 40 mM sodium phosphate buffer (pH 7.0); andstorage in 1.5% human serum albumin in 40 mM sodium phosphate buffer (pH7.0). Values of gene transfer efficiency with the virus relative totransfer efficiency observed for the control after incubation for 7 dayswere calculated and are shown in Table 1.

Gene transfer efficiency was retained at a high level not only using aplate coated with the polypeptide having the heparin-II domain offibronectin (CH-296, CH-271 or H-296) but also using a plate coated withDEAE-dextran or polylysine. It was shown that storage of a retrovirususing such a substance was effective. In addition, the coexistence ofthe substance having a cell-binding activity (C-CS1) did not influencethe storage with the above-mentioned substance.

TABLE 1 1.5% human serum 40 mM sodium albumin in 40 mM Virus phosphatesodium phosphate supernatant buffer buffer CH-296 9.27 54.69 88.58CH-271 12.30 54.57 88.65 H-296 9.91 47.51 83.36 DEAE-dextran 10.96 81.63106.49 DEAE-dextran + 12.77 59.18 101.87 C-CS1 Polylysine 8.54 48.4580.77 Polylysine + 8.42 52.60 81.31 C-CS1 (Values in % are shown in thetable.)

Example 5 Storage of Retrovirus Using Gas-Permeable Culture Bag

CH-296 was used for a 24-well plate without surface treatment (Falcon)or a gas-permeable culture bag (X-FOLD™, 85 cm², Nexell), and thefollowing experiments were carried out.

X-FOLD™ was coated with CH-296 as follows.

First, 9 ml of CH-296 (20 μg/ml) was added to each bag and the bag wasallowed to stand at 4° C. overnight. The bag was then washed three timeswith 30 ml of PBS to obtain a CH-296-coated bag. A 24-well plate wascoated with CH-296 according to the procedure as described in Example 1.

500 μl of a 4-fold dilution of the GaLV/DOG-polII virus supernatant wasadded to each well of a 24-well CH-296-coated plate, and the plate wasincubated in 5% CO₂ incubator at 37° C. for 4 hours to bind theretrovirus vector. 22 ml of the same GaLV/polII virus supernatantdilution was added to X-FOLD™, air was removed from the bag, and the bagwas sealed and incubated in the presence of 5% CO₂ at 37° C. for 4 hoursto bind the retrovirus vector. The containers were washed twice with 40mM sodium phosphate buffer (pH 7.0) or 1.5% human serum albumin in 40 mMsodium phosphate buffer (pH 7.0) (500 μl for the 24-well plate, 30 mlfor the bag). The well or the bag was filled with the same solution; incase of the bag, the bag was sealed after removing air; and the plate orthe bag was incubated at 4° C. for 7 days. In addition, the same virussupernatant dilution was added to the CH-296-coated plate or theCH-296-coated bag; in case of the bag, the bag was sealed after removingair; and the plate or the bag was incubated at 4° C. for 7 days as itwas. On day 7, the solution was removed from the container, a suspensioncontaining K-562 cells at a density of 4×10⁴ cells/ml (500 μl for the24-well plate, 22 ml for the bag) was added, and the plate or the bagwas incubated in the presence of 5% CO₂ at 37° C. for gene transfer. Asa control, a virus-bound container was prepared using a virussupernatant of the same lot and the same procedure, and K-562 cells wereadded immediately without incubation at 4° C. for gene transfer. Valuesof gene transfer efficiency relative to gene transfer efficiencyobserved for the control after incubation for 7 days were calculated.

Gene transfer efficiency of only 10% or less as compared with thecontrol was observed when the retrovirus supernatant dilution was storedin the plate as it was. On the other hand, gene transfer efficiency ofmore than 60% as compared with the control was observed when storage wascarried out in the sealed bag or when the solution was exchanged bywashing the container after binding the retrovirus to CH-296 (the plateor the bag). Based on these results, it was shown that when a retroviruswas stored in the presence of a substance having a retrovirus-bindingactivity, decrease in infectivity of the retrovirus was dramaticallysuppressed by preventing the retrovirus-containing solution fromcontacting air and/or separating the retrovirus from the retrovirusproducer cell supernatant.

Example 6 Long-Term Storage of Retrovirus

A GaLV/DOG-polII virus supernatant (3.3×10⁵ I.V.P./ml) was diluted withRPMI-1640 medium containing 10% fetal calf serum to prepare a 2-folddilution. 500 μl of the dilution was added to each well of a 24-wellCH-296-coated plate prepared as described in Example 1, and the platewas incubated in 5% CO₂ incubator at 37° C. for 4 hours to bind theretrovirus vector. Each well of the plate was washed twice with 500 μlof 40 mM sodium phosphate buffer (pH 7.0) or 1.5% human serum albumin in40 mM sodium phosphate buffer (pH 7.0). The well was filled with thesame buffer, and the plate was incubated at 4° C. In addition, the2-fold dilution of the virus supernatant was added to each well, and theplate was incubated at 4° C. without washing (the virus supernatant wasincubated as it was). The solution was removed from the well 1, 2, 3 or4 week(s) after the initiation of incubation, 500 μl of a suspensioncontaining K-562 cells at a density of 4×10⁴ cells/ml was added, and thecells were cultured in the presence of 5% CO₂ at 37° C. for genetransfer. As to the well incubated with the virus supernatant as it was,the well was washed once with 500 μl of 40 mM sodium phosphate buffer(pH 7.0), and 500 μl of a suspension containing K-562 cells at a densityof 4×10⁴ cells/ml was added, and the cells were cultured in the presenceof 5% CO₂ at 37° C. for gene transfer. As a control, a virus-bound platewas prepared using a virus supernatant of the same lot and the sameprocedure, and K-562 cells were added immediately without incubation at4° C. for gene transfer. Gene transfer efficiency was determined for thecells subjected to gene transfer. Values of gene transfer efficiencywith the virus relative to transfer efficiency observed for the control(i.e., remaining titers) were calculated for the respective test groupsand are shown in Table 2.

TABLE 2 After After After After 1 week 2 weeks 3 weeks 4 weeks 40 mMphosphate buffer 80.46 63.44 47.55 40.76 Human serum albumin in 96.1988.75 73.23 58.93 40 mM phosphate buffer Virus supernatant 56.89 6.242.87 1.98 (Values in % are shown in the table.)

As shown in Table 2, when the virus supernatant was stored as it was,the titer was reduced by one tenth or below after 2 weeks. On the otherhand, when the retrovirus was separated from the culture supernatant(the culture supernatant was replaced by another buffer), 40% or more ofthe titer was retained after storage for 4 weeks. Particularly finestorage stability was observed when a buffer containing a protein (humanserum albumin) was used.

Example 7 Storage of Diluted Retrovirus

A GaLV/DOG-polII virus supernatant (3.3×10⁵ I.V.P./ml) was diluted withRPMI-1640 medium containing 10% fetal calf serum to prepare 2-, 4-, 8-or 16-fold dilutions. 500 μl of the dilution was added to each well of a24-well CH-296-coated plate prepared as described in Example 1, and theplate was incubated in 5% CO₂ incubator at 37° C. for 4 hours to bindthe retrovirus vector. Each well of the plate was washed twice with 500μl of 40 mM sodium phosphate buffer (pH 7.0) or 1.5% human serum albuminin 40 mM sodium phosphate buffer (pH 7.0). The well was filled with 500μl of the same buffer, and the plate was incubated at 4° C. In addition,one of the virus supernatant dilutions was added to each well, and theplate was incubated at 4° C. without washing (the virus supernatant wasincubated as it was). The solution was removed from the well 1, 2, 3 or4 week(s) after the initiation of incubation, 500 μl of a suspensioncontaining K-562 cells at a density of 4×10⁴ cells/ml was added, and thecells were cultured in the presence of 5% CO₂ at 37° C. for genetransfer. As to the well incubated with the virus supernatant as it was,the well was washed once with 500 μl of 40 mM sodium phosphate buffer(pH 7.0), and 500 μl of a suspension containing K-562 cells at a densityof 4×10⁴ cells/ml was added, and the cells were cultured in the presenceof 5% CO₂ at 37° C. for gene transfer. As a control, a virus-bound platewas prepared using a virus supernatant of the same lot and the sameprocedure, and K-562 cells were added immediately without incubation at4° C. for gene transfer. Gene transfer efficiency was determined for thecells subjected to gene transfer. Values of gene transfer efficiencywith the virus relative to transfer efficiency observed for the controlafter storage (i.e., remaining titers) were calculated and are shown inTable 3.

TABLE 3 After 1 week After 2 weeks 40 mM phosphate buffer 2-folddilution 71.91 64.56 4-fold dilution 64.48 50.69 8-fold dilution 59.0643.90 16-fold dilution  58.94 44.28 Human serum albumin in 40 mMphosphate buffer 2-fold dilution 96.24 90.12 4-fold dilution 94.56 85.168-fold dilution 91.76 86.87 16-fold dilution  101.96 99.02 Virussupernatant 2-fold dilution 48.30 8.34 4-fold dilution 38.80 7.35 8-folddilution 29.80 6.65 16-fold dilution  28.24 7.64 (Values in % are shownin the table.)

As shown in Table 3, when the virus supernatant dilution was stored asit was, rapid decrease in titer was observed regardless of the dilutionrate. On the other hand, when the virus supernatant dilution wassubjected to binding to the plate and the culture supernatant was thenreplaced by another buffer, the decrease in titer was remarkablysuppressed. The dilution rate of the virus supernatant used did notconsiderably influence the storage stability of the virus titer.Particularly, when a buffer containing a protein (human serum albumin)was used, influence by the dilution rate was not observed at all. Basedon these results, it was shown that the method of the present inventionwas effective in storage of a retrovirus in a low-titer or diluted virussupernatant.

INDUSTRIAL APPLICABILITY

The present invention provides a method for storing a retrovirus and acomposition containing a retrovirus. According to the storage method, aretrovirus can be stored in a state ready for immediate use forinfection of a cell. Thus, it is useful for studies of retroviruses andin the field of medicine including gene therapy. The composition is alsouseful in the same field.

1. A method for storing a retrovirus, the method comprising maintaininga retrovirus in the presence of a substance having a retrovirus-bindingactivity immobilized on a solid support.
 2. The method according toclaim 1, wherein the retrovirus is maintained in an unfrozen state. 3.The method according to claim 1, wherein the retrovirus is maintained ina solution so that the solution is not in contact with air.
 4. Themethod according to claim 1, wherein the retrovirus is maintained beingseparated from other retrovirus producer cell-derived components.
 5. Themethod according to claim 4, wherein the retrovirus is maintained in abuffer containing a phosphate salt as a buffering component.
 6. Themethod according to claim 4, wherein the retrovirus is maintained in asolution containing a substance selected from the group consisting ofproteins and saccharides.
 7. The method according to claim 1, whereinthe substance having a retrovirus-binding activity is a substanceselected from the group consisting of a polypeptide having theheparin-II domain of fibronectin, fibroblast growth factor, apolypeptide having the insulin-binding domain of type V collagen,DEAE-dextran and polylysine.
 8. A composition containing a retrovirus,wherein the composition is contained in a container carrying a solidsupport on which a substance having a retrovirus-binding activity isimmobilized, and the retrovirus is bound to the substance having aretrovirus-binding activity.
 9. The composition according to claim 8,wherein the retrovirus is contained in a solution in the container sothat the solution is not in contact with air.
 10. The compositionaccording to claim 8, wherein the retrovirus is contained in thecontainer being separated from other retrovirus producer cell-derivedcomponents.
 11. The composition according to claim 10, which furthercontains a buffer containing a phosphate salt as a buffering component.12. The composition according to claim 10, which further contains asubstance selected from the group consisting of proteins and saccharidesin a solution.
 13. The composition according to claim 8, wherein thesubstance having a retrovirus-binding activity is a substance selectedfrom the group consisting of a polypeptide having the heparin-II domainof fibronectin, fibroblast growth factor, a polypeptide having theinsulin-binding domain of type V collagen, DEAE-dextran and polylysine.